1. Field of the Invention
The present invention relates to a protein, a method for immobilizing the protein onto a substrate, a structure including a protein and a substrate, a biosensor including the structure, a nucleic acid encoding the protein, a vector including the nucleic acid, and a kit for detecting a target substance, including the substrate and the protein.
2. Description of the Related Art
Biomolecules typified by nucleic acids and proteins have been known to constitute precise structure. This precise structure is controlled at the atomic level for exerting the functions of the biomolecules. Studies have been made to provide the biomolecules onto a variety of materials by use of such properties of the biomolecules. These techniques have been utilized mainly in fields including biosensors, biomolecule purification processes, and further, the utilization of biomolecules in nanoscale structure formation in semiconductor processes in recent years. For example, Japanese Patent Application Laid-Open No. 2005-312446 discloses a gold-binding protein capable of specifically binding to gold and available in the binding of gold and a substance of interest (target substance). The protein disclosed therein includes at least a portion of an antibody having binding properties to gold. This protein has antibody structure serving as a scaffold and directly recognizes gold based on the antigen recognition ability of the antibody. In Japanese Patent Application Laid-Open No. 2005-312446, this protein is intended to be applied to a biosensor using a gold substrate. Moreover, in Japanese Patent Application Laid-Open No. 2005-312446, the protein is immobilized through molecular recognition toward gold. Therefore, orientation is imparted to the immobilized protein so as to homogeneously immobilize the protein. As a result, improvement in the performance of the biosensor is expected.
However, in Japanese Patent Application Laid-Open No. 2005-312446, molecular recognition, which is seen in antigen-antibody reaction, is utilized in the immobilization of the proteins onto the gold substrate. Therefore, the proteins are noncovalently immobilized thereon. Thus, not a few immobilized proteins might be dissociated. As a result, kinetic analysis is required for biosensor use. Alternatively, application of this protein, when used in a biosensor, is limited to biosensors using a gold substrate. Therefore, a technique for preventing non-specific adsorption to the gold substrate must be devised.
Meanwhile, studies have heretofore been made on a method for immobilizing a protein onto a substrate by use of the physical adsorption or chemical crosslinking of proteins. Alternatively, the production of a biosensor for detecting a variety of target substances usually requires the number of immobilized proteins equal to or more than the number of target substances to be detected. Therefore, an approach for efficiently immobilizing these proteins by a single method is demanded. However, basically, the immobilized proteins are chemically diverse. These immobilized proteins differ in the types and amounts of functional groups and also in the positions of functional groups on the protein surfaces. As a result, it was often very difficult to homogeneously immobilize plural proteins onto a substrate by the same immobilization method.
On the other hand, Langmuir (2002) 18, 2463-2467 discloses a technique for covalently immobilizing proteins onto a substrate via photocrosslinking groups. In this technique, a non-specific adsorption-preventing polymer having photocrosslinking groups is utilized. Therefore, the difference in functional groups depending on protein types is almost negligible. Thus, proteins can be immobilized more homogeneously by this technique than by conventional immobilization. However, Langmuir (2002) 18, 2463-2467 merely suggests that variations in the amounts of proteins immobilized depending on their types are reduced. Langmuir (2002) 18, 2463-2467 avoids any mention of immobilization having the homogeneous orientation of proteins required for biosensors.
Another widely known immobilization method includes introducing a particular sequence (e.g., His Tag or Cysteine residue) into the ends or particular sites of proteins to be immobilized so as to impart enhanced orientation to the proteins thus modified. However, such protein modification might adversely affect the productivity of a certain protein in microorganisms or might reduce the target substance capture activity of the proteins due to the influence of the substrate surface attributed to the low molecular weight of the introduced residue.
The proteins of Japanese Patent Application Laid-Open No. 2005-312446 are immobilized onto a gold substrate through molecular recognition reaction. Therefore, a very homogeneous immobilized state excellent in orientation can be obtained. However, this immobilization, which utilizes only the molecular recognition reaction, is noncovalent on the obtained structure. Therefore, the proteins might be dissociated from the substrate with a certain probability. Thus, Japanese Patent Application Laid-Open No. 2005-312446 provides homogeneous immobilization and, however, does not disclose a method for covalently immobilizing proteins. Moreover, Japanese Patent Application Laid-Open No. 2005-312446 does not disclose a method for immobilizing proteins onto substrates other than the gold substrate by use of molecular recognition reaction.
On the other hand, Langmuir (2002) 18, 2463-2467 discloses the covalent immobilization of proteins via photocrosslinking groups. According to the method of Langmuir (2002) 18, 2463-2467, the difference in functional groups of a variety of proteins is substantially negligible. Thus, proteins can be immobilized homogeneously to some extent onto a substrate. However, Langmuir (2002) 18, 2463-2467 does not disclose an immobilization method capable of imparting orientation to proteins.
Meanwhile, considering the production process, cost and performance of a variety of instruments such as biosensors or diagnostic devices at the commercial level, it is demanded to obtain functions of interest such as sensitivity by use of smaller amounts of capture proteins. It is also demanded to reduce the number of production processes or production time of these instruments. Thus, it is important to efficiently immobilize, in a short time, smaller amounts of proteins with high binding activity to target substances onto a substrate. Alternatively, such a protein immobilization technique is used to produce commercially available, capture protein-mounted medical instruments such as biosensors, diagnostic drugs or diagnostic devices. In this case, it is important to immobilize proteins with more accurate orientation onto a substrate in terms of reproducibility and precision. Protein productivity contributes to the cost reduction of devices. Therefore, it is demanded to produce capture proteins in large amounts in microorganisms.
An object of the present invention is to expand the possibility of providing a technique for satisfying the requirements both for capture proteins and for protein immobilization techniques. Another object of the present invention is to provide a protein capable of crosslinking to a substrate surface through light irradiation. Specifically, this protein can be produced stably even by genetic engineering production in microorganisms and can be immobilized covalently with good orientation onto a substrate.